Project Summary/ Abstract Large endosomes in immune cells, known as phagosomes, contain receptors of diverse functions for pathogen ingestion and inflammatory responses. These receptors cooperate to maximize immune response against infectious agents; their faulty cooperation is implicated in many chronic inflammatory diseases. It is posited that the receptors form clusters to facilitate their synergistic function. However, there is currently no direct evidence supporting this speculation. The critical barrier is the lack of methods to directly measure the functional role of receptor clusters at phagosomes, because phagosomes are intracellular compartments that cannot be directly accessed from the outside once formed. The long-term goal of this project is to elucidate mechanisms by which phagosome receptors cluster and cooperatively regulate the overall immune response. Towards that goal, this R03 pilot study is focused on understanding the clustering and signal crosstalk between Dectin-1 and Toll-like receptor 2 (TLR2) in macrophages. The objective of this proposal is to obtain direct pilot evidence to test the long-standing hypothesis that Dectin-1 and TLR2 cluster at phagosome membranes to cross-regulate the immune response. We will first establish a method that physically decouples receptor clusters and their signaling crosstalk at phagosome membranes. Rather than the homogeneous particles typically used as phagocytic targets, we will use particles with two distinct hemispheres (named Janus particles), each having ligands specific for a particular receptor. Once encapsulated inside phagosomes, the spatial segregation of the two ligands on the particles will force corresponding receptors and signaling proteins to segregate without changing the overall chemical constitution of the phagosome membrane. Changes in cell response produced by this manipulation will reveal the function of receptor clustering. The feasibility of this spatial decoupling strategy has been demonstrated in our preliminary results. In Aim 1, we will employ this approach to identify the mechanism by which Dectin-1 and TLR2 recruit the adaptor protein CARD9 to enable signaling crosstalk. In Aim 2, we will determine the role of Dectin-1 and TLR2 clustering in cross-regulating the macrophage immune responses, by controlling the decoupling and clustering of Dectin-1 and TLR2 on phagosomes. This R03 study will demonstrate the causal relationship between clustering of Dectin-1 and TLR2 and their cooperative regulation of immune response. It will also establish the first method that delineates receptor interactions and signaling through exclusively physical means, in contrast to genetic and pharmacological approaches that are chemical in nature. Because receptor crosstalk in phagosomes is common to many different receptors and various types of immune cells, this spatial decoupling strategy will open the door to an unexplored research area and likely make a broad impact on the field. Pilot results from this R03 proposal will be critical for our future studies that will elucidate the general mechanism of signaling crosstalk in phagosomes.